PROJECT SUMMARY Biofilms are multicellular bacterial communities implicated in the majority of bacterial infections, and nearly all chronic bacterial infections. These communities are nearly impossible to eradicate by traditional chemotherapeutic approaches and represent a major threat to human health. Biofilm bacteria secrete an extracellular matrix (ECM) that limits the ability for phagocytes, complement, antibiotics, and other external stressors to interact with biofilm bacteria. Accordingly, biofilms are highly resistant to antibiotics and the immune system, often necessitating that patients with biofilm-associated infections receive long-term suppressive antibiotics or undergo surgery to remove infected tissues. In addition to inhibiting penetrance of antibiotics and immune defenses, the ECM limits diffusion of nutrients such as oxygen, which, in conjunction with the metabolic activity of resident bacteria, establishes oxygen gradients within biofilms that render the interior of biofilms hypoxic. Several studies have demonstrated that oxygen gradients play a critical role in the development of resilient biofilm communities, and that in biofilms oxygen availability is a central regulator of bacterial metabolism and expression of ECM components. Previous work in uropathogenic Escherichia coli (UPEC), the primary cause of urinary tract infections, has demonstrated that despite being a facultative anaerobe, UPEC relies on aerobic respiration during infection and to form biofilm communities. Through my thesis research, I have shown that UPEC heterogeneously expresses respiratory enzymes, and that these enzymes are expressed in discrete subpopulations. Despite this heterogeneity of expression, only expression of cytochrome bd, a high affinity quinol oxidase necessary for aerobic respiration under hypoxic conditions, is required for UPEC pathogenesis and biofilm formation. Loss of cytochrome bd, but not other quinol oxidases, disrupts biofilm development, alters ECM production, increases susceptibility to antibiotics, and impairs virulence in a murine model of infection. This proposal outlines a series of experiments which will define the role of cytochrome bd in urinary tract infection pathogenesis and the ability for UPEC to form biofilm communities capable of withstanding antibiotic therapy and immune assault. Completion of this proposal will identify bottlenecks that restricts colonization by cytochrome bd deficient UPEC, biochemically define the role of cytochrome bd in the intracellular phase of urinary tract infection, and define mechanisms by which cytochrome bd promotes the formation of antibiotic tolerant biofilms. These studies will yield fundamental insights into how adaptation of central metabolic processes allows bacteria to adapt to diverse host niches and establish resilient biofilm communities, while also investigating cytochrome bd as a potential drug target to aid in the prevention or eradication of biofilm-associated infection.